This application claims priority under 35 USC .sctn. 119 to U.S. application Ser. No. 60/001,365 entitled "Process for Preparing Microspheres Through Phase Inversion Phenomena" filed Jul. 21, 1995 by Edith Mathiowitz, Donald E. Chickering III, Yong S. Jong and Jules S. Jacob.
The application of gene therapy for the treatment of human disease has increased steadily since the first human gene therapy trial was conducted in 1989. To date, more than one hundred gene therapy protocols and clinical trials have been approved by the Recombinant DNA Advisory Committee (RAC) for the treatment of inherited and acquired diseases. Despite the reported advances in gene therapy technology and the increase of approvals in gene therapy protocols, obstacles still remain, including the difficulty in efficient delivery of exogenous genes in vivo.
Gene therapy generally involves the introduction and expression in an animal of an exogenous gene to supplement or replace a defective or missing gene or to produce a product for treating an acquired disease. While there remains some debate about which vectors are most useful under which circumstances, the evolving challenge is not whether gene therapy will work, but rather determining which vectors are the most effective and which delivery schemes are most effective for carrying out gene therapy.
Among the difficulties with delivering exogenous genes to cells for gene therapy is the cell wall itself. Some vectors and naked DNA do not efficiently penetrate mammalian cell walls. This is less of a problem in ex vivo applications where a variety of physio/chemical and mechanical technologies have been developed for introducing genes into cells. Many of these techniques, however, cannot be applied in vivo. Another problem with in vivo delivery of genes to cells is that complex structures such as vectors containing genes under the control of promoters do not fare well in certain physiological environments and are destroyed. These larger complex DNA molecules are unlike short antisense oligonucleotides which typically are modified to protect them against physiological degradation.
The conditions that can destroy an operable gene in vivo are not the only barriers to delivery of genes for gene therapy. The preparative techniques for formulating delivery systems can be destructive to DNA as well. For example, many procedures for forming microparticles require high temperature and/or high sheer forces and/or sonication. Such conditions typically would destroy a vector containing a gene or would result in breakage of a large piece of DNA.
Oral formulations of drugs, although most convenient to the patient, face severe obstacles to delivering the drug molecules to the target cells. This is particularly true for labile drugs such as pieces of DNA or genes. A first obstacle is the stomach. The environment of the stomach is extremely destructive to DNA, and most DNA (and especially large pieces of DNA) would not survive the environment of the stomach. Even if the DNA did survive the environment of the stomach, it then must be taken up by or passed between the cells lining the large and small intestine. Uptake of material across a mucosal epithelial barrier is a selective event, and not all molecules would be expected to be taken up by absorptive and nonadsorptive epithelial cells and/or taken up into systemic circulation. Even if this obstacle is overcome, then the DNA still must resist destruction when in general circulation. The DNA also must gain access accross the membrane of the target cell which is to be transplanted. Finally, the DNA must be presented in a fashion that is nontoxic to the subject. For example, some viral vectors have been shown to induce severe immunological responses in the recipients and some liposomes have been shown to be toxic to recipients.
U.S. Pat. No. 5,075,109, entitled "METHOD OF POTENTIATING AN IMMUNE RESPONSE", issued to Tice, is directed to methods for oral administration of a bioactive agent contained in microparticles to protect the agent from degradation during its passage through the gastrointestinal tract. The patent is particularly directed to a method of oral immunization which will effectively stimulate the mucosal immune system and overcome the problem of degradation of the bioactive ingredient during its passage through the gastrointestinal tract to the Peyer's patch. The '109 patent involves administering bioactive agents contained in microcapsules that are sized between approximately one and ten microns. The microcapsules apparently survive the environment of the stomach and are taken up by the Peyer's patches to stimulate the immune response. The '109 patent does include the term "nucleic acids" as a member of a long list of materials regarded as "bioactive agents". The '109 patent does not mention the delivery of genes, the delivery of genes under the control of a promoter or the delivery of vectors including genes. This is perhaps because the methodology employed by Tice in making the microparticles is typical of prior art fabrication techniques, that is, aggressive emulsification conditions are applied, such as would destroy large pieces of DNA, in order to form the microparticles.
In none of the prior art of which applicants are aware is there disclosed the notion of delivery in microparticles of genes under the control of promoters. Certainly, none disclose the notion of oral delivery of genes under the control of promoters.
It is an object of the invention to provide a noninvasive method of carrying out gene therapy.
Another object of the invention is to provide an oral means of carrying out gene therapy.
Another object of the invention is to provide a method for microencapsulating large pieces of DNA, such as genes under the control of promoters and vectors, in a manner which does not destroy the DNA and that produces a high yield of DNA within the microcapsule. These and other objects are achieved by the present invention.